Plasminogen activator inhibitor type 1 (PAI-1) is the major negative regulator of tissue-type plasminogen activator (tPA-1) and urokinase (uPA) in the fibrinolytic system (Q. Wu et al., Current Drug Targets—Cardiovascular & Haematological Disorders 2 (2002) 27-42). High levels of PAI-1 reduce fibrinolytic potential and can therefore contribute to the development of thrombosis. Thrombotic cardiovascular diseases are particularly evident in elderly populations, but they are also induced in a variety of pathologies associated with aging in general, such as obesity, insulin resistance, diabetes, emotional stress, immune responses, vascular sclerosis or remodeling, and cardiovascular disease generally (K. Yamamoto et al., Cardiovascular Research 66 (2005) 276-285; B. De Taeye et al., Current Opinion in Pharmacology 5 (2005) 149-154).
A number of PAI-1 inhibitors have been reported recently. These include small molecules, antibodies, and peptides (B. Ye et al., Bioorganic & Medicinal Chemistry Letters 14 (2004) 761-765; A. Liang et al., Thrombosis Research 115 (2005) 341-350; C. N. Berry et al., British Journal of Pharmacology 125 (1998) 20-34; J. J. J. van Giezen et al., Thrombosis and Haemostasis 77 (1997) 964-969; P. R. Guzzo et al., Tetrahedron Letters 43 (2002) 41-43).
Despite the clear therapeutic potential of inhibiting PAI-1, enzymes that are capable of achieving this activity in vivo have not heretofore been identified. Subtilisin NAT (nattokinase) was shown recently to cleave PAI-1 in vitro, presumably by virtue of the enzyme's proteolytic activity, but a similar in vivo activity has not been reported (T. Urano et al., Journal of Biological Chemistry 276 (2001) 24690-24696).
There is a need, therefore, for enzymes that can be used in vivo to inhibit PAI-1.